Effect of Temperature on Enzyme Activity

Experiment 4 – Effect of Temperature on Enzyme Activity

Aim
To study the effects of temperature on the activity of amylase enzyme on starch solution.

Introduction
Enzymes are widely known as biological catalyst. Almost all cellular reactions are controlled and guarded by enzymes. Virtually every metabolic reaction which takes place within a living organisms are catalyzed by enzymes. Enzymes are complex three-dimensional globular proteins. Some of the enzymes are built up off proteins and some other associated molecules. Even though enzymes are normal larger than substrates, but only a small part of enzymes molecule actually comes into contact with substrate. This region is called as active site. Enzymes are capable to speed up the metabolic reactions. Enzymes are very specific to its substrate because not all kind of substrate can fit to the enzyme’s active site. The action of enzymes is reversible and enzymes can be reused. This means that an enzyme can catalyze lots of substrates.

Materials
Starch solution, sodium chloride solution, amylase solution, iodine solution

Apparatus
Test tubes, droppers, white tile, stopwatch, water bath controller

Procedure
1. A water bath is set up at 30°C.
2. 3cm3 starch solution added into a test tube labeled A. 1cm3 of sodium solution is added into the test tube A.
3. 2cm3 amylase solution is added to another test tube using a new dropper.
4. Test tubes A and B are immersed in the water bath with temperature which kept constant at 30°C.
5. The test tubes are left for about 5 minutes to allow the solution in test tubes to reach the temperature of the water bath.
6. In meantime, a few drops of iodine are placed on a clean white tile to test the presence of starch.
7. The starch suspension with sodium chloride solution in test tube A is poured into test tube B. The test tube with the mixture is then replaced in the water bath.
8. A stopwatch is activated immediately and the time is recorded as zero minutes.
9. A drop of mixture from the test tube B is immediately placed on the tile containing the iodine solution. Any colour change is observed.
10. The iodine test is repeated at regular intervals of 1/2minutes.
11. The time taken for the hydrolysis of starch to be completed is recorded. The hydrolysis is said was been completed when the mixture no longer turns blue-black in colour when tested with the iodine solution.
12. All the observations are recorded.
13. These procedures are repeated at a temperature of 40°C and 60°C. Volume of solution is kept constant each time.

Observations

Temperature at 30°C
|Interval |

Temperature at 40°C
|Interval |

Temperature 60°C
|Interval |

Overall observation
|Temperature (°C) |Time taken for the hydrolysis of starch to |Rate of enzymatic reaction 1/t (minutes-1) |
| |be completed (minutes) | |
|30 |2.5 |0.40 |
|40 |1.5 |0.67 |
|60 |Not completed |0 |

Discussion
From the experiment, we can conclude that an increase in temperature affects the rate of an enzyme. Controlled reaction in two ways : as the surrounding temperature increases, the rate of reaction is increased until it reaches the optimum temperature. The second way, any increase in temperature, the rate of reaction decrease sharply and the hydrolysis is absolute incomplete at about 60°C.
The explanation for the first way is that, as the temperature increases, or in other words, more heat energy has been supplied, the kinetic energy of the substrate and enzyme molecules increases and thus, the conclusions occur more frequently.
The explanation for the second way is that, beyond the optimum temperature, the heat energy supplied is enough to break the hydrogen bonds and other forces which hold the molecules in their precise shape. This alters the three-dimensional shape of the enzymes and eventually destroys the active site. This means that the substrates can no longer fit into the active sites of the enzymes. Thus, the enzyme is said to be denatured, and loses its catalytic properties. It becomes less soluble and coagulates. Thus, gives the explanation the reason starch is not hydrolyzed at temperature 60°C.
An enzyme is globular and is categorized as tertiary protein. Hydrogen bonding is one of the bonding responsible for shaping the chain. When the heat energy supplied increases, the atoms which make up the enzyme molecules vibrate at faster rate. The more frequent collisions between the substrate and enzyme molecules increase the chances of the substrate molecules coming into contact with the active sites of an enzyme. Active site is a small part of the enzyme molecule which actually comes into contact with the substrate. Thus, the rate of reaction between the substrate and enzyme is increased when the temperature increases until it reaches the optimum temperature which the enzyme catalyses a reaction at the maximum rate. Optimum temperature can be defined at which its reaction rate is the fastest. Theoretically, most enzymes in humans and animals have an optimum temperature at about 37°C.

Conclusion
An increase in temperature affects the activity of salivary amylase on starch. The activity of enzyme is increased when the temperature increases until the optimum temperature has obtained. Beyond the optimum temperature, the rate of enzyme activity decreases as the enzyme has been denatured.

Ways to improve
1. The test tubes containing the mixture are kept in the water bath throughout the experiment. 2. Drops of iodine and starch must be large enough get clearer observation.
3. The dropper used to test the mixture must be washed after test at every interval.

References
1. Larry G. Sellers, David J. Cotter, Susan M. FeldKamp, John Jackson, Larry Lewis, Jan A. Pechenik, Geraldine Ross, Denny Smith, Jane B. Taylor. Biology Concepts and Applications: Instructor’s Resource Manual (2nd Ed), International Thomson Publishing (ITP)
2. Glenn Toole and Susan Toole. (1991), Understanding Biology for Advanced Level (2nd Ed), Stanley Thornes Ltd.
3. William S. Beck, Karel F. Liem, Anne Rose Simpson. (1991). An Introduction to Biology. LIFE (3rd Ed) Harper Collins Publishers.
4. Glenn Toole and Susan Toole (1999), Understanding Biology for Advanced Level (4th Ed), Nelson Thornes Ltd.

References: 1. Larry G. Sellers, David J. Cotter, Susan M. FeldKamp, John Jackson, Larry Lewis, Jan A. Pechenik, Geraldine Ross, Denny Smith, Jane B. Taylor. Biology Concepts and Applications: Instructor’s Resource Manual (2nd Ed), International Thomson Publishing (ITP) 2. Glenn Toole and Susan Toole. (1991), Understanding Biology for Advanced Level (2nd Ed), Stanley Thornes Ltd. 3. William S. Beck, Karel F. Liem, Anne Rose Simpson. (1991). An Introduction to Biology. LIFE (3rd Ed) Harper Collins Publishers. 4. Glenn Toole and Susan Toole (1999), Understanding Biology for Advanced Level (4th Ed), Nelson Thornes Ltd.